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The Open Dermatology Journal, 2010, 4, 10-13
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The Lipid Organisation in Human Stratum Corneum and Model Systems
Joke A. Bouwstra* and Gert S. Gooris
Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA
Leiden, The Netherlands
Abstract: The primary function of the skin is to act as a barrier against unwanted influences from the environment. The
barrier function of the skin is located in the superficial of the skin, the stratum corneum. The stratum corneum consists of
dead cells filled with keratin and water, which are embedded in lipid regions. The lipid regions are the only continuous
structure in the stratum corneum. For this reason the lipid regions are considered to be very important for the barrier function.
The main lipid classes are ceramides, cholesterol and free fatty acids. In this paper the lipid organisation in human stratum
corneum is reviewed. In addition, the role the various lipid classes play in the lipid organisation will be discussed using
mixtures prepared from either native human ceramides or synthetic ceramides. Finally a model, referred to as the stratum
corneum substitute, is described in which the lipid organisation, composition and barrier function can be examined.
Keywords: Lipids, ceramides, X-ray diffraction, lamellar phase, lateral packing, barrier function.
LIPID COMPOSITION
CORNEUM
IN
HUMAN
STRATUM
The upper layer of the skin is formed by a transparent 1015 μm thick layer, which consists of dead cells, corneocytes
embedded in lipid regions. This layer is responsible for the
primary barrier of the skin. The corneocytes are flat dead
cells filled with keratin filaments, water and the natural
moisturising factor [1]. The corneocytes are surrounded by a
densely crosslinked protein layer, referred to as the cell
envelope. A monolayer of lipids is chemically linked to this
densely packed cell envelope [2]. This lipid monolayer has a
crucial role in the stratum corneum as it serves as an
interface between the hydrophilic corneocytes and the
lipophilic extracellular lipid matrix. Furthermore, corneodesmosomes interconnect the corneocytes and play an
important role in the stratum corneum cohesion. The loss of
cells from the stratum corneum is compensated by the cell
growth in the innermost layer of the epidermis, the stratum
basale. In this way the thickness of the epidermis remains
approximately constant.
In human stratum corneum the major lipid classes [3,4]
are ceramides (CERs), cholesterol (CHOL) and saturated
long chain free fatty acids (FFAs). The ratio between these
lipid classes is approximately equimolar [5]. Low levels of
other lipid classes are also present, such as cholesterol
sulphate, glucosylceramides and cholesterol esters. In human
stratum corneum 11 subclasses of CERs have been identified
[6-9]. The CERs head groups are very small and contain
several functional groups that can form lateral hydrogen
bonds with adjacent molecules. The CERs differ by the
head-group architecture and fatty acid chain length. The base
consists of either a sphingosine (S), phytosphingosine (P),
*Address correspondence to this author at the Leiden/Amsterdam Center for
Drug Research, Gorlaeus Laboratories, Leiden University, P.O. Box 9502,
2300 RA Leiden, The Netherlands; Tel: 31-71-5274208; Fax: 31-715274565; E-mail: [email protected]
1874-3722/10
6-hydroxysphingosine (H) or dihydrosphingosine (D).
Chemically linked to the base is a fatty acid, with again three
variations in structure, namely a non-hydroxylated fatty acid
(N), an -hydroxy fatty acid (A) or an -hydroxy fatty acid.
Both, the base and the fatty acid exhibit a chain-length
distribution. Combining the four bases with the N and A
fatty acids results in 8 CER structures, referred to as AS, AP,
AH, AD and NS, NP, NH and ND. The -hydroxy fatty
acids have a very long chain length up to 34 carbon atoms.
Furthermore, a linoleic acid is ester linked to this -hydroxy
group (EO), which results in 3 additional very exceptional
CERs, referred to EOS, EOH and EOP [10].
LIPID ORGANISATION IN
CORNEUM
HUMAN
STRATUM
As far as the lipid organisation is concerned, the lateral
packing as well as the lamellar phases are considered to be
important for the skin barrier function. Already in 1991, the
lamellar phases in human stratum corneum were identified.
Small angle X-ray diffraction studies revealed the presence
of two lamellar phases in human stratum corneum: one
lamellar phase with a periodicity of approximately 6 nm
referred to as SPP (= short periodicity phase), and the long
periodicity phase (LPP) with a periodicity of approximately
13 nm [11] (see Fig. 1). The LPP is present in all species
examined until now, and has a very characteristic molecular
organisation. For this reason it has been suggested that the
presence of this phase plays an important role in skin barrier
function.
When filling a three-dimensional space, the lipid
organisation in the plane perpendicular to the direction in
which the lamellar phases are defined should also be
characterised. In this plane, parallel to the basal plane of the
lamellae, the lipids are arranged in either a liquid phase, a
hexagonal phase or an orthorhombic lateral packing. The
orthorhombic lateral packing is very densely packed. The
neighboring molecular distance is not equal in all directions.
2010 Bentham Open
The Lipid Organisation in Human Stratum Corneum and Model Systems
The Open Dermatology Journal, 2010, Volume 4
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Fig. (1). A schematic drawing of the lamellar phases in human stratum corneum. Between the corneocytes the lipids are organised in two
lamellar phases with repeat distances of 6 and 13 nm. However, these lamellar phases fill the three dimensional space only in one direction.
In the plane perpendicular to the lamellar phases, another organisation can be identified, namely the lateral packing. This lateral packing is
either liquid, hexagonal or orthorhombic. The orthorhombic lateral packing is dominantly present in human stratum corneum.
In one direction, the lipids are more densely packed,
resulting in two strong diffraction rings at 0.37 and 0.41 nm
spacing (see Fig. 1).
Wide angle X-ray diffraction studies revealed the
presence of an orthorhombic lateral packing in human
stratum corneum [12-14]. However, whether a hexagonal
phase coexists with the orthorhombic lateral packing
remained unclear, as the characteristic 0.41 nm spacing
characteristic for the hexagonal phase is at the same position
as the strong intensity ring in the diffraction patterns of the
orthorhombic lateral packing. However, using electron
diffraction single crystals are exposed to the electron beam,
which makes it easier to distinguish the two phases. Electron
diffraction revealed the presence of an hexagonal lateral
packing [15, 16]. Studies performed as function of depth in
stratum corneum showed that the hexagonal lateral packing
is more prominently present in the top layers than in the
deeper layers of human stratum corneum. The presence of an
orthorhombic phase is also observed with infrared
spectroscopy and indicates that the lipids within the lamellae
are very densely packed [17]. Very recently it has been
shown that there is a correlation in the degree the
orthorhombic lateral packing is present in stratum corneum
and the trans-epidermal water loss values [18]. The latter is a
measure for the skin barrier. This demonstrates that the
orthorhombic lateral packing plays indeed a role in the skin
barrier function. It remained unclear whether a liquid phase
coexisted with the orthorhombic lateral packing, as the broad
reflection of the liquid phase in the diffraction pattern was
obscured by the reflections based on soft keratin present in
the corneocytes. Frequently some CHOL phase separates
from the lamellar phases [12, 13].
RELATION BETWEEN LIPID COMPOSITION AND
ORGANISATION
Information on the relationship between lipid
organization and lipid composition is of great importance to
unravel the mechanism controlling the skin barrier function
of normal and diseased skin. Due to the complexity of the
native stratum corneum, it is impossible to modulate
systematically lipid composition in the stratum corneum.
Especially when one is interested in the relationship between
lipid composition and lipid organisation, the use of lipid
mixtures isolated from stratum corneum, offers an attractive
alternative. In this paper we will focus on the phase
behaviour studies performed with human CERs.
When using isolated human CERs and CHOL only, the
SPP and LPP with periodicities of 5.4 and 12.8 nm
respectively, were formed in the lipid mixtures mimicking
the lamellar phases in human stratum corneum already very
closely (see Fig. 2) [19]. This demonstrates that no proteins
are required for the formation of these lamellar phases and
that in the absence of FFA, the LPP is already formed. The
lamellar phases remained unchanged over a wide range of
12 The Open Dermatology Journal, 2010, Volume 4
CHOL:CER molar ratios (between 0.2 and 1). This indicates
that the formation of the lamellar phases is insensitive
towards changes in CHOL:CER molar ratio. At a
CHOL:CER molar ratio higher than 0.6, the presence of
crystalline CHOL is observed suggesting that the lamellae
are already saturated with CHOL at this molar ratio. This
demonstrates that in the in vivo situation a variation in
CHOL:CER molar ratio will not lead to a substantial change
in lipid phase behavior, but that CHOL will form separate
crystalline domains when exceeding the amount necessary to
saturate the lamellae. When focusing on the lateral packing,
an orthorhombic packing is only formed in the presence of
FFA. Furthermore, in the presence of FFA the LPP and SPP
were present in the lipid mixtures with repeat distances of
13.0 and 5.5 nm, respectively, demonstrating that
CHOL:CER:FFA mixture mimics closely the lipid
organisation in intact human stratum corneum [19, 20].
When using short chain FFA with predominantly C16 and
C18 chain length, no orthorhombic lateral packing was
detected in CHOL:CER:FFA mixtures [21]. In the presence
of long-chain FFAs, besides an orthorhombic also a liquid
packing was formed in the lipid mixtures, as the broad
reflection at 0.46 nm (indicative for a liquid phase) was
clearly observed in the diffraction pattern.
Fig. (2). The relationship between lipid composition and lipid
organisation. CHOL and CER are very crucial for the formation of
the two lamellar phases, while FFA induce the formation of an
orthorhombic lateral packing.
In diseased or dry skin often the lipid composition is
different from that in healthy subjects. For example, the
levels of EOS and NP are both reduced in stratum corneum
of psoriasis skin [22], while the fatty acid levels in stratum
corneum of lamellar ichthyosis skin is drastically diminished
[23]. Consequently, the lipid organisation may also change:
phase studies with lipid mixtures demonstrated that a
reduction in EOS level reduces the formation of the LPP,
while a reduction in FFA will decrease the population of
lipids forming an orthorhombic lateral packing. These
findings correlated excellently with ex vivo studies. In
stratum corneum of lamellar ichthyosis patients the
formation of an orthorhombic lateral packing is strongly
reduced, while a reduction in EOS level in normal and dry
stratum corneum is correlated with a reduction in the
formation of the LPP [16, 24]. These results demonstrate that
lipid mixtures prepared with isolated CER are an excellent
tool to provide information on the relation between lipid
Bouwstra and Gooris
organisation and composition relevant for the changes in
lipid organisation of diseased skin.
LIPID MIXTURES BASED ON SYNTHETIC CERs
In contrast to the previous investigation, in which solely
mixtures prepared with isolated CERs with a variation in
chain length were studied, in a more recent study [25] the
lipid organisation in mixtures prepared with synthetic CERs
with defined acyl chain length was examined. The CER
included in these mixtures are EOS (C30), NS (C24), NP
(C16 and C24), AS (C24) and AP (C24). The lipid
organisation in equimolar mixtures of CHOL, synthetic
CERs and FFAs closely resembles that in stratum corneum,
as both LPP (12.2. nm) and SPP (5.4 nm) are present and the
lateral packing of the lipids is orthorhombic. Interestingly, in
these mixtures FFAs are required for proper lipid
organisation, as only in their presence a dominant formation
of the LPP could be detected. No data are available yet,
whether in these mixtures CER and FFA participate in one
orthorhombic lattice. This is of interest as a reduction in
chain length variation often reduces the mixing properties.
Very interestingly, the mixing properties of the synthetic
CERs, CHOL, FFA mixtures mimicked closely that of
mixtures prepared from isolated CERs, most probably due to
the fatty acid chain length variation [26].
As it is of interest not only to study the relation between
lipid composition and organisation, but also the effect of
lipid composition on the barrier function, a stratum corneum
lipid membrane has been developed mimicking very closely
the lipid organisation and orientation in stratum corneum.
This lipid membrane is supported by a porous membrane,
and is referred to as the stratum corneum substitute (SCS).
The SCS is designed in such a way that after preparation it
can be fixed in a flow-through diffusion cell to study its
barrier properties (see Fig. 3A). These properties were
evaluated in a series of in vitro passive diffusion studies,
using three structurally related compounds, namely paminobenzoic acid (PABA), ethyl-PABA and butyl-PABA.
PABA is the most hydrophilic compound and its
lipophilicity increases with increasing ester chain length. The
diffusion profiles of all three model compounds across 12
m thick lipid membranes closely resemble those of human
stratum corneum [27]. Exclusion of EOS from the lipid
mixture revealed a reduced barrier function of the stratum
corneum substitute, demonstrating that CER1 is not only
very important for a proper skin lipid phase behaviour, but
also for the skin barrier function (see Fig. 3B). This indicates
that the reduced barrier function in psoriatic and LI skin
might partially be caused by a reduction in EOS in stratum
corneum. In future we will use this excellent tool to study the
relation between lipid organisation and lipid composition
into more details to determine the role the various lipids play
in the skin barrier function.
CONCLUSIONS
Although impressive progress has been made in
elucidating the lipid organisation in stratum corneum,
essential information is still missing. For example the lipid
lamellar organisation is still under debate as the localisation
of the lipids within the LPP and SPP is not fully understood.
Furthermore, only limited information is available on the
The Lipid Organisation in Human Stratum Corneum and Model Systems
lipid organisation and composition in stratum corneum of
diseased skin. However, this unequivocally stresses that
there is a need to explore this scientific field in more detail.
The Open Dermatology Journal, 2010, Volume 4
[7]
[8]
[9]
[10]
[11]
[12]
[13]
Fig. (3A). Diffusion studies can be performed across a lipid
membrane mimicking the barrier properties and lipid organisation
of human stratum corneum. These membranes are referred to as the
stratum corneum substitute.
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
Fig. (3B). In the absence of EOS, (referred to as CER1) an increase
in the flux of ethyl-PABA is observed, indicating a reduced barrier
function.
[22]
[23]
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© Bouwstra and Gooris; Licensee Bentham Open.
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